A Highlights from MBoC Selection: Regulation of microtubule-based transport by MAP4

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.     

Vertebrate melanophores as potential model for drug discovery and development: A review

Drug discovery in skin pharmacotherapy is an enormous, continually expanding field. Researchers are developing novel and sensitive pharmaceutical products and drugs that target specific receptors to elicit concerted and appropriate responses. The pigment-bearing cells called melanophores have a significant contribution to make in this field. Melanophores, which contain the dark brown or black pigment melanin, constitute an important class of chromatophores. They are highly specialized in the bidirectional and coordinated translocation of pigment granules when given an appropriate stimulus. The pigment granules can be stimulated to undergo rapid dispersion throughout the melanophores, making the cell appear dark, or to aggregate at the center, making the cell appear light. The major signals involved in pigment transport within the melanophores are dependent on a special class of cell surface receptors called G-protein-coupled receptors (GPCRs). Many of these receptors of adrenaline, acetylcholine, histamine, serotonin, endothelin and melatonin have been found on melanophores. They are believed to have clinical relevance to skin-related ailments and therefore have become targets for high throughput screening projects. The selective screening of these receptors requires the recognition of particular ligands, agonists and antagonists and the characterization of their effects on pigment motility within the cells. The mechanism of skin pigmentation is incredibly intricate, but it would be a considerable step forward to unravel its underlying physiological mechanism. This would provide an experimental basis for new pharmacotherapies for dermatological anomalies. The discernible stimuli that can trigger a variety of intracellular signals affecting pigment granule movement primarily include neurotransmitters and hormones. This review focuses on the role of the hormone and neurotransmitter signals involved in pigment movement in terms of the pharmacology of the specific receptors.     

Effect of the barring gene on eye pigmentation in the fowl

Pigment cells of the iris, pecten, retinal pigment epithelium, and choroid of the wild-type jungle fowl (JF) and the barred Plymouth rock (BPR) breeds of adult chickens were studied at both light and electron microscopic levels. BPR choroidal tissues had 2.8 times fewer melanophores than the JF choroid, and BPR melanophores also contained 2.4 times fewer melanosomes, which tended to clump together in variously sized clusters. The melanosomes were often irregular in shape, smaller in diameter, and less mature (stage III) than those granules in the JF. The retinal pigment epithelium of both JF and BPR breeds contained a single epithelial layer of columnar cells. Rod-shaped melanosomes were present in the more apical regions of this cell type in both breeds. Both JF and BPR irides contained a multilayered posterior pigmented epithelium of columnar shaped cells that were densely filled with large spherical granules. Intercellular spaces with interdigitating cytoplasmic projections were present between pigment cells of both breeds. The pecten melanophores of both breeds were dendritic with melanosomes that were larger and fewer in numbers than those pigment cells of the iris and choroid. Intercellular spaces were present between cells in both breeds, with numerous villous-like pigment cell extensions. Choroid melanophores contained very little, if any, acid phosphatase activity. Approximately one-half of the retinal pigment epithelial cells observed contained small amounts of diffuse acid phosphatase activity in both breeds. The iris and pecten melanophores of both breeds contained profuse acid phosphatase activity scattered throughout their cytoplasms. Sparse tyrosinase activity was seen in iris and pecten pigment cells, whereas no tyrosine activity was observed in choroid melanophores or in retinal pigment epithelial cells in the two breeds, indicating that little new melanogenesis occurs in adult pigmented eye tissues. The results show that the barring gene reduces the number and melanin content of the choroidal melanophores in homozygous male BPR chickens as compared to the wild-type JF chickens. Whether this gene prevents the initial migration of embryonic neural crest cells (future melanophores) to the choroid or whether some of the choroidal melanophores prematurely degenerate in the embryo of young birds is yet to be determined. If the latter is the case, this choroid system may serve as a model for a genetic hypomelanotic disease such as vitiligo.     

Regulation of Melanogenesis in Melanocytes

Melanogenesis refers to the biosynthesis of melanin pigment in pigment cells called melanocytes. Melanins are mixed biopolymers formed during a series of oxidation/reduction reactions that are initiated by the enzymatic hydroxylation of L-tyrosine to L-dopa. In living cells, melanogenesis is limited to melanosomes, the membrane bounded microscopic secretory granules of melanocytes. Melanosomes may be secreted into the environment as, for example, from the squid's ink gland; or be transferred to neighboring cells, such as the keratinocytes in human skin and hair; or they may remain within the pigment cell and change only their subcellular localization, as in the rapidly color-changing dermis of lower vertebrates. Regulation of the melanocytic phenotype involves synthesis of the biosynthetically active subcellular apparatus of melanogenesis, premelanosomes and tyrosinase, and the utilization of the final product, melanized melanosomes, in the translocation and secretory processes mentioned above. Genetic information for this regulation is stored in the nuclear genome whose expression is controlled by the intra- and extracellular environment. As premelanosomes become biosynthetically active, they mature into melanosomes by fusing with vesicles derived from the trans-Golgi network and the plasmalemma, thereby internalizing and incorporating contents and membrane components from inside the cell and the cell surface. In the process, melanosomes become acidified. The thesis pursued in this review explores the importance of the melanosome as the final common pathway of regulation of melanin biosynthesis.     

Chromatophoromas in a pine snake

Iridophoroma and melanophoroma were diagnosed in an adult male pine snake. Light microscopic examination of irregularly thickened white and black portions of abnormal scales demonstrated two distinctive populations of pigment-containing cells. Pigment cells within abnormal-appearing white scales had needle-shaped granules that were dark amber in color while black portions were composed of pigment cells typical of melanophores, with dark black, round granules. Both populations of cells showed junctional activity, and clusters of both neoplastic pigment cell types were found in adjoining areas of the epidermis. By electron microscopy, the pigment cell with amber-colored granules contained reflecting platelet profiles typical of iridophores while pigment cells with dark round granules contained melanosomes. At a junctional area between abnormal white and black scales, mosaic chromatophores containing reflecting platelet profiles and melanosomes were observed. At 1 1/2 years following initial diagnosis, the snake died and neoplastic iridophores were found at multiple visceral sites; there was no evidence of metastases of melanophores to any organ. The two pigment cell tumors are believed to have developed from either stem cells destined to become iridophores and melanophores or from prexisting iridophores and melanophores in the dermis.     

Dynamics of the redistribution of pigment granules in the dermal melanophores of anuran amphibians. 1. Dispersion

The dispersion of melanosomes in the dermal melanophores of the Xenopus laevis larvae has been studied by time--lapse cinematography. The process began with the appearance of distally directed melanosome flows in the cell cytoplasm. During the subsequent migration of pigment granules, the flows branched forming branches of the 2nd and higher orders. The whole cytoplasm became filled with a layer of melanosomes. During the dispersion, the movement of melanosomes in a flow is replaced by their dispersion all over the cytoplasm; these processes alternated. In the peripheral part of the cell devoid of melanosomes, membrane vesicles appeared and the cytoplasm was distinctly divided into ecto- and endoplasm. The ectoplasm contained numerous microfilaments and single microtubules, the endoplasm did not contain any cell organelles, except single electron-dense melanosomes. The active role of plasma membrane in the intracellular movement of melanin granules is suggested.     

Opposite effect of lipofuscin granules and melanosomes from human retinal pigment epithelium of eye on photooxidation of cardiolipin

The influence of lipofuscin granules and melanosomes from human retinal pigment epithelium on the light-induced photooxidation of cardiolipin liposomes and the generation of superoxide radicals was studied. Lipofuscin granules were able to stimulate, while melanosomes inhibited, the cardiolipin photooxidation. The visible light irradiation of both melanosomes and lipofuscin granules generated superoxide radicals with mean rates of 1.5 nmole/min/10(7) and 38 nmole/min/10(7) granules, accordingly. However, melanosomes but not lipofuscin granules reacted readily with superoxide radicals. Moreover, the rate constant of degradation of superoxide radicals in the presence of melanosomes was about five orders of magnitude higher than the rate constant of its photogeneration. Therefore, we propose that melanosomes in retinal pigment epithelium cells have a photoprotective role whereas lipofuscin granules may stimulate photodestructive reactions.     

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.     

Inhibition of melanosome transformation in embryonic chick pigment retina cultured in vitro.

When the retinal pigment epithelial cells of the chick embryo are transferred to monolayer cultures, they lose their phenotypic trait-- melanin granules-- after a few days. Within the first 24 hours almost all of the melanosomes and premelanosomes are transformed into the degradative structures of the dense bodies or the melanosome complexes. Then, within a few days, these structures disappear completely from the cytoplasm. Actinomycin D, added to the culture medium during the first four hours, almost completely prevents the transformation of melanosomes and premelanosomes. The inhibition of cell proliferation, caused by the addition of colcemid, does not prevent the transformation, though the time of initiation of transformation is delayed considerably. The mechanisms of the transformation of pigment granules are discussed.     

Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells

In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.     

Biogenesis of melanosomes in Kupffer cells of Proteus anguinus (Urodela,Amphibia)

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single-membrane-bounded bodies, named as 'clusters of melanosomes' or 'melanosomogenesis centers'. Inside such clusters, different structures are present: (1) filament-like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron-dense melanosomes in different stages of melanization, (3) melanosomes with an electron-dense cortical area and a less electron-dense medullar area, and (4) uniformly highly electron-dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.     

Identification of two types of melanocyte within the stria vascularis of the mouse inner ear

We have distinguished two types of melanocyte within the intermediate layer of the stria vascularis in the cochlea of normally pigmented mice: light and dark intermediate cells. The light intermediate cells are present in the stria from birth and have the typical appearance of a melanocyte. They are large and dendritic with electron-lucent cytoplasm containing numerous vesicles that show tyrosinase activity, and pigment granules in various stages of development. These granules have the ultrastructural and histochemical characteristics of premelanosomes and melanosomes. The light intermediate cells persist throughout life, but less frequently contain pigment in older animals. The dark intermediate cells, present only in adult mice, vary considerably in number and distribution between animals. Pigment granules, bound within an electron-dense acid phosphatase-rich matrix, form the main component of the dark intermediate cells. The intermediate cells may comprise either two distinct cell populations or different developmental stages of the same cell type; ultrastructural observations suggest the latter. In young mice, light intermediate cells contain the electron-dense matrices, which at later stages of development are found almost exclusively in dark cells. The dark intermediate cells contain few cell organelles other than pigment granules accumulated within lysosomal bodies and they often have pycnotic nuclei. These observations suggest that the dark intermediate cells are a degenerate form of the light intermediate cells. Clusters of melanosomes also occur in the basal cells, and to a much lesser extent in the marginal cells. These cells do not stain after incubation in DOPA, suggesting that they are not capable of melanin synthesis, and therefore probably acquire melanin by donation from adjacent melanocytes. Pigment clusters are also found within the spiral ligament at all stages of development.     

Stimulation of Distinct D2 Dopaminergic and α2-Adrenergic Receptors Induces Light-Adaptive Pigment Dispersion in Teleost Retinal Pigment Epithelium

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules aggregate and disperse in response to changes in light conditions. Pigment granules aggregate into the RPE cell body in the dark and disperse into the long apical projections in the light. Pigment granule movement retains its light sensitivity in vitro only if RPE is explanted together with neural retina. In the absence of retina, RPE pigment granules no longer move in response to light onset or offset. Using a preparation of mechanically isolated fragments of RPE from green sunfish, Lepomis cyanellus, we investigated the effects of catecholamines on pigment migration. We report here that 3,4-dihydoxyphenylethylamine (dopamine) and clonidine each mimic the effect of light in vivo by inducing pigment granule dispersion. Dopamine had a half-maximal effect at approximately 2 nM; clonidine, at 1 microM. Dopamine-induced dispersion was inhibited by the D2 dopaminergic antagonist sulpiride but not by D1 or alpha-adrenergic antagonists. Furthermore, a D2 dopaminergic agonist (LY 171555) but not a D1 dopaminergic agonist (SKF 38393) mimicked the effect of dopamine. Clonidine-induced dispersion was inhibited by the alpha 2-adrenergic antagonist yohimbine but not by sulpiride. These results suggest that teleost RPE cells possess distinct D2 dopaminergic and alpha 2-adrenergic receptors, and that stimulation of either receptor type is sufficient to induce pigment granule dispersion. In addition, forskolin, an activator of adenylate cyclase, induced pigment granule movement in the opposite direction, i.e., dark-adaptive pigment aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long-term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore-fibroblast co-culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast-derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane-enclosed melanosomes in a process that was upregulated by alpha-melanocyte-stimulating hormone (alpha-MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane-proteins seemed to be of importance, as the cluster-like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long-term colour changes and for studies of factors that affect pigmentation.     

Ultimate fate of rod outer segments in the retinal pigment epithelium.

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

Loss of melanin by eye retinal pigment epithelium cells is associated with its oxidative destruction in melanolipofuscin granules

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.     

Ultimate Fate of Rod Outer Segments in the Retinal Pigment Epithelium

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

Biogenesis of Melanosomes in Kupffer Cells of Proteus anguinus (Urodela,Amphibia)

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single‐membrane‐bounded bodies, named as ‘clusters of melanosomes’ or ‘melanosomogenesis centers’. Inside such clusters, different structures are present: (1) filament‐like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron‐dense melanosomes in different stages of melanization, (3) melanosomes with an electron‐dense cortical area and a less electron‐dense medullar area, and (4) uniformly highly electron‐dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.     

Stimulation of the CLIP-170--dependent capture of membrane organelles by microtubules through fine tuning of microtubule assembly dynamics

Cytoplasmic microtubules (MTs) continuously grow and shorten at their free plus ends, a behavior that allows them to capture membrane organelles destined for MT minus end-directed transport. In Xenopus melanophores, the capture of pigment granules (melanosomes) involves the +TIP CLIP-170, which is enriched at growing MT plus ends. Here we used Xenopus melanophores to test whether signals that stimulate minus end MT transport also enhance CLIP-170-dependent binding of melanosomes to MT tips. We found that these signals significantly (>twofold) increased the number of growing MT plus ends and their density at the cell periphery, thereby enhancing the likelihood of interaction with dispersed melanosomes. Computational simulations showed that local and global increases in the density of CLIP-170-decorated MT plus ends could reduce the half-time of melanosome aggregation by ~50%. We conclude that pigment granule aggregation signals in melanophores stimulate MT minus end-directed transport by the increasing number of growing MT plus ends decorated with CLIP-170 and redistributing these ends to more efficiently capture melanosomes throughout the cytoplasm.     

Fine Structure of Melanogenesis in the Ink Sac of Sepia offidnalis

The ink sac epithelium of the cuttlefish Sepia officinalis was investigated by electron microscopy. Melanogenesis in a simplified view seems to follow the general scheme of melanin formation in vertebrates. First, a membrane-bound protein matrix is formed, which is called an early stage melanosome. The early stage melanosomes are more or less irregular in shape with a size up to 1.5 μm and contain membranous, granular, or vesicular material. They seem to originate from Golgi bodies and/or endoplasmic reticulum. Membranes that frequently are present in the early stage melanosomes may originate from fusion of vesicles or from incorporation of Golgi membranes into early stage melanosomes. Free cytoplasmic material or mitochondria probably are also incorporated into the early stage melanosomes or melanosomes. Therefore, the origin of the early stage melanosomes seems to be similar to that of autophagosomes. The early stage melanosomes mature to melanosomes in which several dozen melanin granules are formed. These melanosomes, at last, release the melanin granules together with other cellular material, including early stage melanosomes, into the lumen of the ink gland. This finding confirms the earlier postulated holocrine character of the release. Active tyrosinase was localized in the lumen of the ink sac as already shown by biochemical methods. There was also additional evidence that most of the material of broken down cells inside the lumen of the ink sac seems to be converted into melanin granules.